Differentiation Potential Of Wharton’ Jelly Derived Mesenchymal Stem Cells Into Cardiomyocytes

Objective:This study through the umbilical cord Wharton jelly source of mesenchymal stem cells in vitro induced by transplantation and in vivo to observe the to the characteristics of myocardial cell differentiation, myocardial reconstruction new excellent seed cells and in the in vivo and in vitro molecular, cell, and the overall level, confirmed Wharton’jelly source of mesenchymal stem cells to cardiac differentiation potential, WJMSCs repair and reconstruction of myocardial clinical research lay a theoretical basis. Methods:Extracting Wharton’ jelly derived mesenchymal stem cells, which cut into small tissue blocks, it was used to isolate mesenchymal stem cells by stick wall method, after tissue adherent culture for 12 to 14 days,which the cell fusion for primary cells, when the growth of adherent cells reached 80-90% confluence subculture. Second generations of well growing cells were detected CD73, CD90, CD105, HLA-ABC, CD34,, CD45, HLA-DR, and CD44 by flow cytometry. Cell proliferation was detected by using four methyl salt(MTT) method, and the second generation of WJMSCs was used to identify the growth of bone, fat and cartilage differentiation.Cardiac specific differentiation gene expression feature detection, which application QT-PCR quantitative detection of WJMSCs myocardial early specific transcription factor Isl-1, Flk-1, Nkx2.5 expression; by 5-azacytidine induced differentiation into cardiomyocytes and myocardial cell specific marker a-actin, TNT, GATA-4 and connexin-43.Making rat model of acute myocardial infarction(AMI), In the model establishment of 14 days in vivo WJMSCs transplantation, graft volume is 0.5ml(2 x 106 cells), myocardial infarction rat transplantation WJMSCs a month, by the ultrasound echocardiography examination and observe the changes of cardiac function; immunofluorescence confocal, m RNA and protein levels of myocardial cell specific a-actin, TNT, GATA-4, connexin-43. Results:Umbilical cord Wharton jelly by tissue adherent culture, 6 days WJMSCs from the tissue blocks climbed out of the, spindle shaped adherent growth, 12-14 days WJMSCs around the tissue block growth and close to the fusion, after 24 h after passage, the cells entered the logarithmic growth phase, 6 days later, cells into the platform growth period, slowing proliferation. Eight days later, the cells into decline phase, from the second generation of growth good WJMSCs for flow cytometric detection of surface markers and WJMSCs expression of CD44, CD73, CD90, CD105, HLA-ABC, did not express CD34, CD45 and HLA-DR, in accordance with the characteristics of mesenchymal stem cells.WJMSCs in bone, fat, cartilage induction medium cultured for 2 weeks, cell alkaline phosphatase, alizarin red, oil red "O", methylene blue staining showed positive reaction, indicating that WJMSCs to osteoblast, adipocyte, chondrocyte differentiation. Quantitative detection of QT-PCR WJMSCs early cardiac specific transcription factor Flk-1, Isl-1, Nkx2.5 expression; WJMSCs after induced by 5-azacytidine can differentiate into cardiomyocyte like cells, and the expression of a-actin m RNA in myocardial cell specific markers, TNT, GATA-4, connexin-43.The rats by ligating the anterior descending after ligation of large area myocardial surface pale, and accompanied by ventricular wall motion disordered or weakened, electrocardiogram shows two or more than two adjacent lead Q wave shape and wave width > 1/4R, width> 0.04 s, left ventricular ejection fraction <45%, cardiac muscle infarction the model rats were made successfully; after cell transplantation by echocardiography, LVEF transplantation group(61.62 + 8.74%) after treatment than before treatment(43.02 + 3.67%) and the control group after treatment(43.43 + 4.2%) was significantly higher than(P<0.01); LAVW transplantation group after LAVW treatment(1.84 + 0.36 mm) than before treatment(1.28 + 0.45 mm) and the control group after treatment(1.21 + 0.31 mm) significantly higher than(P<0.5); confocal immunofluorescence, m RNA and protein level of myocardial cell specific markers-actin, Tn T, GATA-4 alpha, connexin-43 detection, WJM SCs transplantation group was significantly higher than that of the control group. Conclusions: Studies have shown that WJMSCs in vitro culture with the potential of multi-directional differentiation, with transcription factors in early stage of myocardial cells, by 5-azacytidine could be induced to differentiate into myocardial cells and expression of myocardial cell specific cell surface marker, confirmed that the WJMSCs in vitro can differentiated into myocardial cells; WJMSCs after in vitro transplantation into infarcted myocardium in rats in vivo, can significantly improve cardiac function in rats, the pathological examination of cardiac specific markers were significantly higher than control group. In experimental rat myocardial infarction model, the WJMSCs in vivo can be differentiated into myocardial cells.